The principle of confocal laser scan microscopy for two-dimensional, quantitative fluorescence measurement is illustrated in FIGS. 1 and 2. FIG. 1 shows the optical setup of a 2-D flying spot confocal laser scan microscope, using for fluorescence excitation a laser beam 11, a dichroic beam splitter 12, a 2-dimensional scan engine 13 for spatial beam deflection in two orthogonal directions (X-Y) and a lens 14 for focusing the laser beam into an object plane 15. Fluorescent light of a longer wavelength than the excitation laser 11 is generated by exciting fluorescent molecules in the object plane 15.
Fluorescent light emitted by fluorophores located in the object plane 15 of the scanned area is collected by lens 14 and then transmitted by means of the scan engine 13 and the dichroic beam splitter 12 as a fluorescent light beam 17 which is focused by lens 18 into a pinhole aperture 19 in a conjugate plane 21 in front of a photodetection device 22.
The concept of confocal imaging, which is currently used to discriminate the generally weak fluorescence signal from background radiation, is illustrated in FIG. 2. Only optical radiation from within the confocal volume Vc, i.e., the fluorescence signal, is detected by the photodetector 22. Vc is defined by the optical transfer function of the detection optics (OTFem) and the size of the detector pinhole 19 in the conjugate plane 21. Higher background suppression rates result for smaller confocal volumes Vc.
The size of the scan field of view is typically in the order of 20×20 square millimeter. The confocal volume is generally in the order of Vc=5×5×50 cubic micrometer, where Ac=5×5 square micrometer and zc=50 micrometer is approximately the spot size and the Rayleigh range of the focused laser beam, respectively. The pixel size of the scan engine 13 for scanning the laser beam 11 in the field of view is typically 1 to 20 micrometer.
DNA binding arrays, e.g. those of the type described in U.S. Pat. No. 5,143,854, consist of a glass chip carrying a chemical system subdivided in adjacent cells, commonly called features. The features are characterized by specific probes. Specific nucleic acid sequences are immobilized (captured) by the probes and labeled with a fluorescent dye. The amount of captured nucleic acid on individual features is detected using quantitative fluorescence measurement (the fluorescent dye emits light when excited by light energy of a given wavelength) by sequential pixel reading (scanning) of the features. The features are spatially over-sampled by the scanning procedure (i.e. number of pixels>number of features) for accurate spatial referencing of the glass chip by numerical data analysis and for increased feature signal quality by averaging physically measured light intensities. Typical pixel sizes are in the order of 1 to 20 micrometer.
The ratio of the scan field of view to the cross-section Ac of the confocal volume is typically high in confocal laser scan microscopy, i.e. “scan field of view”/“cross-section Ac of the confocal volume”>>1, which readily leads to a x-y position depending optical transfer function OTF(x, y)=OTFex*OTFem, where OTFex and OTFem are the optical transfer functions of the excitation and emission optics, respectively. The x-y position dependence is mainly due to mechanical misalignment and imperfections of optical and opto-mechanical components, such as e.g. the scan engine used for scanning. It causes an inhomogeneous sensitivity over the scan field of view, as schematically sketched in
FIGS. 3a, 3b and 3c and this in turn leads to erroneous quantitative fluorescence measurements. As an example, FIG. 4 schematically shows the scanned image of a DNA binding array, e.g. of the type described in U.S. Pat. No. 5,143,854, which array has a chess-board pattern. As described hereinafter with reference to FIG. 4 the scanned image has a lower signal level in the top right corner, due to either inhomogeneous fluorophore density in the scanned object or inhomogeneous sensitivity of the confocal laser scan microscope over the scan field of view.
There is therefore a need for a reliable quantitative measurement and evaluation of the sensitivity over the scan field of view of a confocal laser scan microscope of the above described type.
The availability of an appropriate reference standard target object would allow to discriminate between instrument- and scanned object (e.g. a DNA binding array of the type described in U.S. Pat. No. 5,143,854) contributions to the observed non-uniformity in FIG. 4. However, no reference fluorescing target objects for characterizing key performances of a confocal laser scan microscope, i.e., sensitivity, limit of detection, uniformity-, spatial resolution- and signal dynamic behavior over the scan field of view, have been reported yet.
There is therefore a need for an appropriate reference standard target object that allows one to discriminate between instrument- and scanned object contributions to a non-uniformity of the type represented in FIG. 4.